Method for preventing or delaying the onset of diabetes

ABSTRACT

A method for preventing, delaying the onset of or treating diabetes in a patient comprising selecting a patient who is susceptible to developing diabetes, who is developing diabetes or who is diabetic and administering to the patient one or more than one dose of a pharmaceutical agent comprising a polynucleotide encoding a secreted exogenous protein, such as a secreted luciferase or a secreted form of human glutamic acid decarboxylase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/257,221, titled “Method of Delaying the Onset of Diabetes”and filed Oct. 7, 2002, which is a national phase application ofPCT/US01/12392, titled “Method of Preventing or Treating Diabetes,”filed Apr. 17, 2001, which claims the benefit of U.S. patent application60/198,137, titled “Method for Preventing Diabetes,” filed Apr. 17,2000; the contents of which are incorporated herein by reference intheir entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support underCooperative Agreement Number DAMD17-97-2-7016 with the National MedicalTechnology Testbed, Inc., United States Department of the Army. TheUnited States Government has certain rights in this invention.

BACKGROUND

Diabetes is a major cause of morbidity and mortality in the UnitedStates and throughout the world. Diabetes is a metabolic diseasecharacterized by the inability to metabolize glucose and generallydivided into two types. Of the two types, type 1 diabetes usuallyresults from autoimmune destruction of beta cells in the pancreas duringadolescence which leads to insufficient insulin production.

Research into the causes and treatments for type 1 diabetes frequentlyinvolve the use of nonhuman animals. The non-obese diabetic (NOD) mouseis one animal model system generally accepted for studying type 1diabetes as NOD mice develop a form of diabetes that parallels type 1diabetes in humans, including sharing common susceptibility factors suchas major histocompatibility complex molecules. Studies of NOD mice andhumans have indicated that two proteins synthesized by pancreatic betacells play determining roles as autoantigens responsible for the onsetof diabetes. The two proteins are the hormone insulin, a secretedprotein, and the enzyme glutamic acid decarboxylase (GAD), anintracellular protein found as either soluble GAD67, or membrane-boundGAD65 in beta cells. The importance of these two autoantigens fordiabetes onset in NOD mice is indicated by the finding that mostpathogenic CD8+ T cells recognize a single insulin epitope, and thatmice with beta cell-specific reduced expression of gad65/67 genes do notdevelop diabetes. In humans, the presence of anti-insulin and anti-GADautoantibodies has been used to predict the onset of diabetes. Thereremains, however, a need for a method of preventing diabetes in humans.

SUMMARY

According to the present invention, there is provided a method forpreventing, delaying the onset of or treating diabetes in a patient. Themethod comprises, first, selecting a patient who is susceptible todeveloping diabetes, who is developing diabetes or who is diabetic.Next, the patient is administered one or more than one dose of apharmaceutical agent comprising a polynucleotide encoding a secretedexogenous protein.

In a preferred embodiment, selecting the patient comprises identifyingin the patient the presence of anti-insulin or anti-GAD autoantibodies,or identifying in the patient the presence of increasing hyperglycemia,or identifying in the patient the presence of glycosuria, or identifyingin the patient the presence of a genetic predisposition to diabetes. Ina particularly preferred embodiment, the one or more than one dose is aplurality of doses. In another particularly preferred embodiment,administering to the patient one or more than one dose comprisesinjecting the patient intramuscularly with the one or more than onedose. In another preferred embodiment, the method comprises, afteradministering, monitoring the patient for the development diabetes.

In a particularly preferred embodiment, the exogenous protein is asecreted Renilla luciferase comprising a sequence according to SEQ IDNO:1 or according to SEQ ID NO:3.

FIGURES

These and other features, aspects and advantages of the presentinvention will become better understood with regard to the followingdescription, appended claims, and the accompanying figure where:

FIG. 1 is graph showing the percent on non-diabetic, cyclophosphamidetreated NOD mice given versus time where the NOD mice were injected withplasmid DNA encoding full-length human GAD65 (filled boxes); SGAD55, asecreted form of human GAD65 (open boxes); SRUC3, a secreted form of theRenilla reniformis luciferase (open circles); or no injections ofplasmid DNA (filled circles).

DESCRIPTION

According to one embodiment of the present invention, there is provideda method for delaying the onset of type 1 diabetes or preventing type 1diabetes in a human or animal. The method comprises selecting a patientwho is susceptible to developing diabetes or who is developing diabetesand administering to the patient one or more than one dose of apharmaceutical agent. The pharmaceutical agent can comprise apolynucleotide encoding a secreted Renilla luciferase or can comprise, apolynucleotide encoding a secreted form of glutamic acid decarboxylase.

The present invention resulted from an investigation ofcyclophosphamide-accelerated diabetes in non-obese diabetic (NOD) micethat were administered plasmid DNA encoding either intracellular humanGAD, a secreted form of human GAD or a secreted form of Renillareniformis luciferase. In summary, four-week old animals were injectedwith plasmid DNA encoding either intracellular human GAD, a secretedform of human GAD or a secreted form of Renilla reniformis luciferase.Animals injected with plasmid DNA encoding secreted GAD demonstrated asignificant reduction in the incidence of diabetes. Animals injectedwith plasmid DNA encoding Renilla reniformis luciferase demonstrated asignificant delay in the onset of diabetes. However, animals injectedwith plasmid DNA encoding intracellular GAD did not demonstrate either asignificant reduction in the incidence of diabetes or a significantdelay in the onset of diabetes, even though previous studies showed thatinjecting plasmid DNA encoding human GAD65 or a secreted GAD preventedislets inflammation (insulitis) in the pancreas of NOD mice. The presentinvention will now be disclosed in detail.

First, plasmids designated pND2-SRUC3, pND2-GAD65 and pND2-SGAD55 wereconstructed as disclosed in Liu, J. et al. (1999). Intramuscularinjection of plasmid DNA encoding intracellular or secreted glutamicacid decarboxylase causes decreased insulitis in the non-obese diabeticmouse. Gene Ther. Mol. Biol. 3, 197-206. The cDNAs carried by theplasmids were respectively; sruc3, SEQ ID NO:1, encoding a secreted formof the soft coral Renilla reniformis luciferase; gad65, SEQ ID NO:2,encoding full-length human GAD65 protein; and sgad55, SEQ ID NO:3, amodified gad65 cDNA encoding a secreted truncated form of human GAD65,SEQ ID NO:2, that had an 88 amino acid amino-terminal region deletionremoving a palmitoylation sequence and a Golgi targeting signal thatprevents secretion of full-length GAD65 protein, leaving residues265-1758 of SEQ ID NO:2. The deleted sequence was replaced with a humanIL-2 sequence encoding a signal peptide, which was cleavedintracellularly before secretion. This truncated GAD65 protein (GAD55)contained all known epitopes recognized by antibodies from patientssuffering from type 1 diabetes. Each cDNA was placed undertranscriptional control of the cytomegalovirus promoter in plasmid pND2.In addition, each plasmid carried a CoL-E1 origin of replication and agene encoding ampicillin resistance for amplification of plasmid DNA inthe bacterium Escherichia coli.

The plasmid DNA was amplified in E. coli strain DH5-a, and isolatedusing the alkaline-lysis method followed by standard double-round cesiumchloride purification. DNA quality and quantity were determined with aU.V. spectrophotometer (A₂₆₀/A₂₈₀ ratio greater than 1.8), and agarosegel electrophoresis. The plasmid DNA was then dissolved under sterileconditions in phosphate buffer saline (PBS) at a final concentration of2 μg/μl, and stored at −20° C.

The plasmid DNA was administered intramuscularly to female NOD/MrkTacmice (Taconic Laboratories, Germantown, N.Y. US). Each animal wasinjected into each quadriceps muscle with 200 micrograms/leg at the ageof four weeks using disposable tuberculin syringes fitted with 27Gneedle, and an identical set of injections two days later, for a totalof 800 micrograms plasmid DNA/mouse. Three groups of mice receivedinjections of pND-2 plasmid carrying either sruc3, SEQ ID NO:1, gad65,SEQ ID NO:2, or sgad55, SEQ ID NO:3, cDNA. An additional group of micewas left untreated as control. All mice received intra-peritonealinjections of cyclophosphamide (200 mg/kg) at the age of 10 and 12 weeksto accelerate the onset of diabetes. The mice were kept in an animalfacility under non-pathogen free conditions, and received injection ofDNA under general anaesthesia using 66 mg/kg body weight ketamine(Phoenix Scientific, St Joseph, Mo. US), 7.5 mg/kg body weight oxylazine(Lloyd Laboratories, Shenandoa, 10 US), and 1.5 mg/kg body weightacepromazine maleate (Fermenta Animal Health Co., MO US).

The onset of diabetes in the animals was determined by monitoring forglycosuria twice a week with Clinistix Reagent Strips for urine analysis(Bayer Corporation, Elkhart, Ind. US). Once glycosuria was present, adiagnosis of diabetes was confirmed when blood glucose levels weregreater than 300 mg/deciliter on two consecutive days (using Accu-Chek™Advantage (Roche Diagnostics Corporation, Indianapolis, Ind. US).Animals were sacrificed when diagnosed as diabetic, or at the end of theobservation period when the animals were 18 weeks old. Statisticalanalysis of the results were performed using a Kaplan-Meier analysiswith a log-rank and Mann-Whitney test to detect differences inprevention and delay of diabetes onset.

Referring now to FIG. 1, there is shown a graph depicting the results ofadministering the DNA given as the percent on non-diabetic,cyclophosphamide treated NOD mice given versus time, where the NOD micewere injected with plasmid DNA encoding full-length human GAD65 (filledboxes); SGAD55, a secreted form of human GAD65 (open boxes); SRUC3, asecreted form of the Renilla reniformis luciferase (open circles); or noinjections of plasmid DNA (filled circles). As can be seen, thepercentage of 18-week old animals that remained non-diabetic afterreceiving injection of plasmid pND2-SRUC3, pND2-GAD65 or pND2-SGAD55 was18, 30 and 63, respectively, and 23 percent of untreated animals werefree of diabetes at the end of the observation period. A Kaplan Meierplot of diabetes onset, together with statistical analysis, indicatedthat the mice which received injections of pND2-SGAD55 had astatistically significant reduction in diabetes when compared withuntreated control (P=0.05, log-rank test). By contrast, mice thatreceived injection of pND2-GAD65 did not show a statisticallysignificant reduction in diabetes when compared with the same controls(P=0.37, log rank test). Although groups of untreated mice and mice thatreceived injection of PND2-SRUC3 had a similar percentage ofnon-diabetic animals at the end of the observation period, injection ofpND2-SRUC3 resulted in a significant delay in the onset of diabetes whencompared to untreated controls using the Mann-Whitney test (P=0.01).Differences between the three groups of treated mice with respect toonset were not found to be statistically significant (P>0.13, log ranktest).

Anti-luciferase and anti-GAD IgG and IgG1 antibody levels in mice serawere determined using ELISA to detect an immune response to luciferaseand GAD polypeptides after injection of pND2-SRUC3, pND2-GAD65, andpND2-SGAD55 plasmids as follows. Blood was collected (0.5-1.5 ml) afterheart puncture, and sera were obtained from samples after twocentrifugations at 3,000×g for 10 minutes at 4° C., and 10 ml of 1%sodium azide was added to each sample. Ninety-six well microtiter plates(Dynex Technologies Inc., Chantilly, Va. US) were coated overnight at 4°C. with 100 ml phosphate buffer saline (PBS) containing 5 mM DTT, 100 mMbeta-mercaptoethanol, and 10 mg/ml recombinant human GAD55 proteinisolated from E. coli, or BSA. After blocking with 0.5% BSA in PBS for 2hours at 37° C., serially diluted sera were added to wells and allowedto incubate for 2 hours at 37° C. Unbound proteins and antibodies wereremoved with four washes of PBS+0.1% Triton x100 for 5 minutes at roomtemperature after each reaction. Alkaline phosphatase-conjugatedFab-specific anti-mouse IgG monoclonal antibodies (Sigma, St Louis, Mo.US) were diluted 1:40,000 in blocking buffer, added to wells, andincubated for 2 hours at 37° C. The relative amounts of bound antibodieswere determined after addition of 100 ml alkaline phosphatase substrateLumi-Phos Plus (Lumigen Inc., Southfield, Mich. US) to each well. Lightemission catalyzed by alkaline phosphatase was measured in a ML3000Luminometer (Dynex Technologies Inc., Chantilly, Va. US) after allowingthe reaction to develop for 30 minutes at 37° C.

No increase in anti-luciferase IgG was detected in sera of mice thatreceived injections of pND2-SRUC3 plasmid DNA, when compared with theother three groups of NOD mice and with a control group of untreated CD1mice. Further, there were no apparent differences in the range of titersof anti-GAD IgG antibodies in sera of mice that received injections ofpND2-GAD65 and pND2-SGAD55 plasmids, when compared with untreatedcontrols and with mice that had received injection of plasmidpND2-SRUC3. However, titers of anti-GAD IgG were in generally higher inNOD mice than in CD1 controls, in contrast with titers ofanti-luciferase IgG.

Because Th2 cells mediate a process that leads to production of IgG1antibodies, IgG1 levels were determined and were used as a marker ofwhether a Th2 type of response was induced. IgG1 antibodies weredetermined using a corresponding protocol to that above, except thatalkaline phosphatase-conjugated IgG1-specific anti-mouse IgG monoclonalantibodies (Zymed Laboratories Inc., South San Francisco, Calif. US)were used for detection at a dilution of 1:2,000.

No increase in anti-luciferase IgG1 titer was detected in sera of micethat received injections of plasmid pND2-SRUC3, when compared to othergroups of NOD mice and with CD1 controls. Similarly, there was noincrease in anti-GAD IgG1 titer in sera of NOD mice from the group thatreceived injection of plasmid pND2-SRUC3 and the untreated group whencompared to CD1 mice. By contrast, however, an increase in anti-GAD IgG1titer was detected in several non-diabetic mice that received injectionsof plasmid pND2-SGAD55. In addition, some of the mice that receivedinjection of plasmid pND2-GAD65 also showed increased anti-GAD IgG1titer, but the increase did not always correlate with an absence ofdiabetes. None of the animals had titer of anti-luciferase and anti-GADIgG2a antibodies above background.

These results indicate that insulitis scores obtained previously, Liu,J. et al., after injection of plasmid DNA encoding SRUC3, which did notdecrease insulitis, and GAD65, which did decrease insulitis, were notpredictive of diabetes prevention. Further, cellular location of humanGAD polypeptide encoded by a genetic vaccine affects prevention ofCYP-accelerated diabetes in the NOD mouse because injection of plasmidDNA encoding full-length intracellular GAD did not significantly preventdiabetes, while injection of plasmid encoding secreted GAD did decreasediabetes frequency. The decrease was accompanied by an increase inanti-GAD IgG1 titers, implicating the involvement of Th2 lymphocytes. Inaddition, the finding that both the injection of DNA encoding secretedforeign protein and secreted autoantigen delays the onset of diabetesimplies that secretion of exogenous protein alone effects disease onset.

In one embodiment, the present invention is a method of preventing,delaying the onset of or treating diabetes in a patient. The methodcomprises, first, selecting a patient who is susceptible to developingdiabetes, who is developing diabetes or who is diabetic. The selectioncan be made using standard methods as will be understood by those withskill in the art with reference to this disclosure. For example, theselection can be made by identifying in the patient the presence ofanti-insulin or anti-GAD autoantibodies or both anti-insulin and oranti-GAD autoantibodies, the presence of increasing hyperglycemia, thepatient the presence of glycosuria, the presence of a geneticpredisposition to diabetes or more than one of these.

Next, the patient is administered one or more than one dose of apharmaceutical agent comprising a polynucleotide encoding a secretedexogenous protein. In a preferred embodiment, the pharmaceutical agentis administered in a plurality of doses. In another preferredembodiment, the dose is between about 0.001 mg/Kg and about 10 mg/Kg. Inanother preferred embodiment, the dose is between about 0.01 mg/Kg andabout 1 mg/Kg. In another preferred embodiment, the dose is about 0.1mg/Kg. In another preferred embodiment, the dose is administered weeklybetween about 2 and about 10 times. In a particularly preferredembodiment, the dose is administered weekly 4 times.

In a particularly preferred embodiment, the secreted exogenous proteinis a secreted Renilla luciferase comprising a sequence according to SEQID NO:1. In another particularly preferred embodiment, is a secretedform of human glutamic acid decarboxylase comprising a sequenceaccording to SEQ ID NO:3. Additionally, the method can comprise, afteradministering, monitoring the patient for the development diabetes.

EXAMPLE 1

According to the present invention, the onset of diabetes in a patientis delayed or prevented, for example, as follows. First, the patient isselected based on the presence of circulating anti-insulin and anti-GADautoantibodies. Next, the patient is injected intramuscularly with 0.1mg/Kg of a pharmaceutical agent comprising a plasmid encoding a secretedform of human glutamic acid decarboxylase. The injection is repeatedweekly for 3 weeks while the level of circulating anti-insulin andanti-GAD autoantibodies is monitored. The treatment is ended when thelevel of circulating anti-insulin and anti-GAD autoantibodies hasreturned to normal.

All references cited in this disclosure are incorporated herein byreference in their entirety. Although the present invention has beendiscussed in considerable detail with reference to certain preferredembodiments, other embodiments are possible. Therefore, the scope of theappended claims should not be limited to the description of preferredembodiments contained in this disclosure.

1. A method for preventing or delaying the onset of diabetes in apatient comprising: a) selecting a patient who is susceptible todeveloping diabetes, who is developing diabetes or who is diabetic; b)administering to the patient one or more than one dose of apharmaceutical agent comprising a polynucleotide encoding a secretedglutamic acid decarboxylase.
 2. The method of claim 1, where selectingthe patient comprises identifying in the patient the presence ofanti-insulin or anti-GAD autoantibodies or both anti-insulin andanti-GAD autoantibodies.
 3. The method of claim 1, where selecting thepatient comprises identifying in the patient the presence of increasinghyperglycemia.
 4. The method of claim 1, where selecting the patientcomprises identifying in the patient the presence of glycosuria.
 5. Themethod of claim 1, where selecting the patient comprises identifying inthe patient the presence of a genetic predisposition to diabetes.
 6. Themethod of claim 1, where the one or more than one dose is a plurality ofdoses.
 7. The method of claim 1, where administering to the patient oneor more than one dose comprises injecting the patient intramuscularlywith the one or more than one dose.
 8. The method of claim 1, where theglutamic acid decarboxylase comprises a sequence according to SEQ IDNO:3.
 9. The method of claim 1, further comprising, after administering,monitoring the patient for the development diabetes.